Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist.

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.

Molecular cloning and functional expression of the mouse CRF2(a) receptor splice variant.

The mouse corticotropin-releasing factor (CRF) type 2a receptor (CRF2(a)) splice variant was cloned by a PCR-based approach. The corresponding cDNA was found to encode a 411-amino acid polypeptide with highest sequence homology to the rat CRF2(a) receptor. By semiquantitative reverse transcriptase PCR (RT-PCR) analysis, the CRF2(b) mRNA was mainly found in the heart and skeletal muscle with only low level expression in the brain. In contrast, CRF2(a) mRNA was restricted to the brain with major expression sites in the cortex, hippocampus, hypothalamus and telencephalon. Binding and cyclic AMP stimulation studies showed a similar ligand selective profile for both mCRF2 receptor splice variants. A notable exception however, was urotensin I which displayed a approximately 3-fold higher affinity for the CRF2(a) receptor and also stimulated cyclic AMP production in mCRF2(a)-transfected cells with a approximately 3-fold higher potency than in mCRF2(b)-transfected cells. These data show that the mouse like other mammalian species expresses two ligand-selective CRF2 receptor splice variants and that the mCRF2(a) receptor is the predominant central CRF2 receptor in the mouse.

Five amino acids of the Xenopus laevis CRF (corticotropin-releasing factor) type 2 receptor mediate differential binding of CRF ligands in comparison with its human counterpart.

The ligand selectivity of human (hCRF(2A)) and Xenopus laevis (xCRF(2)) forms of the corticotropin-releasing factor type 2 (CRF(2)) receptor differs. The purpose of this study was to identify amino acids in these two CRF(2) receptors conferring these differences. An amino acid triplet in the third extracellular domain (Asp(262)Leu(263)Val(264) in hCRF(2A) or Lys(264)Tyr(265)Ile(266) in xCRF(2)) was found to diverge between both receptors. When binding and signaling characteristics of receptor mutants hR2KYI and xR2DLV were assessed, the tri-amino acid motif replacement produced receptors with binding properties resembling the xCRF(2) receptor. The converse mutation created a mutant receptor with a binding pharmacology identical to the profile of the hCRF(2A) receptor. This effect was most notable for xR2DLV, which possessed a binding affinity for astressin approximately 15-fold greater for astressin than sauvagine. In contrast, the binding profiles of the hCRF(2A) receptor and hR2KYI did not differ. These data indicate that another domain of the xCRF(2) receptor mediated low-affinity binding of astressin. Two amino acids in the first extracellular domain differ in xCRF(2) (Asp(69)Ser(70)) and hCRF(2A) (Glu(66)Tyr(67)) receptors. The hCRF(2A) receptor mutant (hR2DS-KYI) bound astressin with a low affinity indistinguishable from the xCRF(2) receptor. Therefore, these data demonstrate that ligand selectivity differences between amphibian and human forms of the CRF(2A) receptor are governed by these two motifs of the extracellular domains of the xCRF(2) receptor.